Silks represent an important advanced biomaterial due to their unique and impressive mechanical properties, versatility in processing to control structure and morphology, degradability, biocompatibility and genetic tailorability. While silk has a long term historical use in sutures, recent studies have expanded both fundamental and applied aspects of this protein in many areas related to advanced biomaterials. The hypothesis for this proposal is that silk protein bioengineering, with appropriate consideration for processing and chemical modifications, can be employed to modulate the rate of 3D scaffold degradation and tissue-specific outcomes suitable for bone growth and repair. Reprocessed native silkworm silk formed into 3D scaffold porous structures, in combination with cytokine and adhesion sites, will be used in perfusion bioreactor systems and in vivo rat models. Our goal is to understand the Irelationships between silk scaffold structure and function related primarily to degradation and bone-like tissue Ioutcomes in vitro and in vivo. The outcome of the proposed study will be an understanding of fundamental Irelationships between silk structure and morphology with function (mechanical properties, rates of degradation, cellular responses related to bone tissue formation). The outcome of the proposed studies will be a roadmap of the limpact of processing and modification factors on silk structure and function for this family of advanced biomaterials. IIn part, by mimicking the native processing of silk, we envision a range of possible outcomes in terms of structures /and functions based on silk scaffolds that will provide useful interactions with cells toward bone-specific needs. The /three specific aims will address: (a) Relationships between silk crystallinity and porosity in 3D scaffolds on mechanical properties and rates of enzyme degradation in vitro. (b) How scaffold structure, morphology and surface chemistry (RGD, BMP-2 decoration) impact bone-like tissue formation from Saos-2 (human osteosarcoma cells) and MSCs (human adult bone marrow stem cells). Cellular and genetic assessments of bone-related outcomes, spatial and temporal deposition of calcium using non-destructive imaging methods (mCT), and changes in structural, mechanical and morphology features of the scaffolds will be assessed. (c) Biocompatibility of the matrices in vitro and in vivo. The data from the proposed study will improve the understanding and utility of silk-based advanced biomaterials for a wider range of biomedical applications.